Whole-Genome Sequencing of Two Potentially Allelopathic Strains of Bacillus from the Roots of C. equisetifolia and Identification of Genes Related to Synthesis of Secondary Metabolites.

The coastal Casuarina equisetifolia is the most common tree species in Hainan's coastal protection forests. Sequencing the genomes of its allelopathic endophytes can allow the protective effects of these bacteria to be effectively implemented in protected forests. The goal of this study was to sequence the whole genomes of the endophytes Bacillus amyloliquefaciens and Bacillus aryabhattai isolated from C. equisetifolia root tissues. The results showed that the genome sizes of B. amyloliquefaciens and B. aryabhattai were 3.854 Mb and 5.508 Mb, respectively. The two strains shared 2514 common gene families while having 1055 and 2406 distinct gene families, respectively. The two strains had 283 and 298 allelochemical synthesis-associated genes, respectively, 255 of which were shared by both strains and 28 and 43 of which were unique to each strain, respectively. The genes were putatively involved in 11 functional pathways, including secondary metabolite biosynthesis, terpene carbon skeleton biosynthesis, biosynthesis of ubiquinone and other terpene quinones, tropane/piperidine and piperidine alkaloids biosynthesis, and phenylpropanoid biosynthesis. NQO1 and entC are known to be involved in the biosynthesis of ubiquinone and other terpenoid quinones, and rfbC/rmlC, rfbA/rmlA/rffH, and rfbB/rmlB/rffG are involved in the biosynthesis of polyketide glycan units. Among the B. aryabhattai-specific allelochemical synthesis-related genes, STE24 is involved in terpene carbon skeleton production, atzF and gdhA in arginine biosynthesis, and TYR in isoquinoline alkaloid biosynthesis. B. amyloliquefaciens and B. aryabhattai share the genes aspB, yhdR, trpA, trpB, and GGPS, which are known to be involved in the synthesis of carotenoids, indole, momilactones, and other allelochemicals. Additionally, these bacteria are involved in allelochemical synthesis via routes such as polyketide sugar unit biosynthesis and isoquinoline alkaloid biosynthesis. This study sheds light on the genetic basis of allelopathy in Bacillus strains associated with C. equisetifolia, highlighting the possible use of these bacteria in sustainable agricultural strategies for weed management and crop protection.

Inhibition of co-occurring weeds and young sugarcane seedling growth by perennial sugarcane root extract.

Allelopathy is a process whereby a plant directly or indirectly promotes or inhibits growth of surrounding plants. Perennial sugarcane root extracts from various years significantly inhibited Bidens pilosa, Digitaria sanguinalis, sugarcane stem seedlings, and sugarcane tissue-cultured seedlings (P < 0.05), with maximum respective allelopathies of - 0.60, - 0.62, - 0.20, and - 0.29. Allelopathy increased with increasing concentrations for the same-year root extract, and inhibitory effects of the neutral, acidic, and alkaline components of perennial sugarcane root extract from different years were significantly stronger than those of the control for sugarcane stem seedlings (P < 0.05). The results suggest that allelopathic effects of perennial sugarcane root extract vary yearly, acids, esters and phenols could be a main reason for the allelopathic autotoxicity of sugarcane ratoons and depend on the type and content of allelochemicals present, and that allelopathy is influenced by other environmental factors within the rhizosphere such as the presence of old perennial sugarcane roots. This may be a crucial factor contributing to the decline of perennial sugarcane root health.

Is allelochemical synthesis in Casuarina equisetifolia plantation related to litter microorganisms?

Productivity decline of Casuarina equisetifolia plantation and difficulty in natural regeneration remains a serious problem because of allelopathy. Previous studies have confirmed that 2,4-di-tert-butylphenol (2,4-DTBP) are the major allelochemicals of the C. equisetifolia litter exudates. The production of these allelochemicals may derive from decomposition of litter or from the litter endophyte and microorganisms adhering to litter surfaces. In the present study, we aimed to evaluate the correlation between allelochemicals in litter and endophytic and epiphytic fungi and bacteria from litter. A total of 100 fungi and 116 bacteria were isolated from the interior and surface of litter of different forest ages (young, half-mature, and mature plantation). Results showed that the fermentation broth of fungal genera Mycosphaerella sp. and Pestalotiopsis sp., and bacterial genera Bacillus amyloliquefaciens, Burkholderia-Paraburkholderia, and Pantoea ananatis had the strongest allelopathic effect on C. equisetifolia seeds. Allelochemicals, such as 2,4-DTBP and its analogs were identified in the fermentation broths of these microorganisms using GC/MS analysis. These results indicate that endophytic and epiphytic fungi and bacteria in litters are involved in the synthesis of allelochemicals of C. equisetifolia. To further determine the abundance of the allelopathic fungi and bacteria, Illumina MiSeq high-throughput sequencing was performed. The results showed that bacterial genera with strong allelopathic potential were mainly distributed in the young and half-mature plantation with low abundance, while the abundance of fungal genera Mycosphaerella sp. and Pestalotiopsis sp. were higher in the young and mature plantations. In particular, the abundance of Mycosphaerella sp. in the young and mature plantations were 501.20% and 192.63% higher than in the half-mature plantation, respectively. Overall, our study demonstrates that the litter fungi with higher abundance in the young and mature plantation were involved in the synthesis of the allelochemical 2,4-DTBP of C. equisetifolia. This finding may be important for understanding the relationship between autotoxicity and microorganism and clarifying the natural regeneration problem of C. equisetifolia.

Response mechanisms of sugarcane seedlings to the allelopathic effects of root aqueous extracts from sugarcane ratoons of different ages.

Sugarcane ratoon performance declines with increasing age due to the intergenerational accumulation and release of allelochemicals by old sugarcane roots. We aimed to clarify the effects of these allelochemicals on sugarcane seedling growth under continuous sugarcane ratoon cropping. We investigated the allelopathic effects of treatment with root aqueous extracts from sugarcane ratoons of different ages on the osmoregulatory substance content, antioxidant enzyme activity, membrane lipid peroxidation product content, photosynthetic physiological characteristics, and root exudate enzymatic characteristics of sugarcane seedlings. The root aqueous extracts exerted allelopathic effects on sugarcane seedlings. The relative electrolyte leakage, proline content, soluble protein content, soluble sugar content, malondialdehyde content, and catalase activity of the leaves were more sensitive than those of the roots. Conversely, the superoxide dismutase and peroxidase activities of the roots (positive response) were more sensitive than those of the leaves (negative response). The total chlorophyll content and net photosynthetic rate of the leaves exhibited relatively sensitive negative responses. Urease activity negatively responded but sucrase activity positively responded. We concluded that the root aqueous extracts of sugarcane ratoons may exert their allelopathic effects by affecting the level of osmoregulatory substances and causing peroxidative damage to the cell membranes of sugarcane seedlings and altering the activities of various enzymes in the rhizosphere.

Structural variability and niche differentiation of the rhizosphere and endosphere fungal microbiome of Casuarina equisetifolia at different ages.

BACKGROUND: Casuarina equisetifolia is one of the most important artificially planted protective forests along the coast in southern China for windbreaks, soil erosion, and sand dune stabilization. Self-renewing of C. equisetifolia is very limited, which might be caused by low soil nutrient levels and reduced microbial activity. METHODS: Use of high-throughput sequencing of the 18S rDNA to investigate the microbial communities from the rhizosphere and root endosphere of C. equisetifolia in young-aged, intermediate-aged, and mature-aged forests. RESULTS: Our results indicate that the diversity of rhizosphere fungal microbiomes in field-grown C. equisetifolia is much lower than that of the endosphere microbiomes. Bioinformatic analysis showed that rhizocompartments produce the strongest differentiation of rhizosphere and endosphere communities. Notably, the distribution of rhizosphere fungi communities was significantly influenced by the environmental factors, not by forest ages. CONCLUSIONS: The presented study suggests that the rhizocompartments and environmental factors, rather than forest ages, determine the diversities of fungal community.

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Litter plays an important role in ecosystems. To compare the diversity and community structure of microorganisms inside and outside litter, Casuarina equisetifolia were selected from three forests in Guilin coastal area of Haikou City, Hainan Province. Illumina Miseq high-throughput sequencing was used to analyze the diversity and composition of epiphytic and endophytic bacteria of litter. The results showed that the diversity of epiphytic bacteria was higher than that of endophytic bacteria. Moreover, the diversity and richness of bacteria inside and outside C. equisetifolia litter in the intermediate-aged forest were the highest, followed by young forest and mature forest. Proteobacteria and Actinobacteria were the most abundant at the phylum level, accounting for about 80% of the total. At the genus level, Curtobacterium, Jatrophihabitans, Mycobacterium, Actinomycetospora, Mucilaginibacter and Pseudomonas showed significant variation among different forest ages. PCoA results showed that the endophytic bacteria of litter were greatly affected by forest age, while the epiphytic bacteria were greatly affected by environmental factors. The fermentation broth of Bacillus amyloliquefaciens had the strongest allelopathic potential to C. equisetifolia seeds and 2,2'-methylenebis (6-tert-butyl-4-methyl-phenol) was found in it, indicating that B. amyloliquefaciens was involved in the synthesis of allelochemicals. The diversity of litter microbial community affected the allelopathy of C. equisetifolia, which laid a foundation for studying the role of microorganisms in the degradation process of C. equisetifolia litter.